The highly conserved SAGA complex plays important roles in gene regulation in yeast, fly, mouse, and human cells. Acetyltransferase activity in SAGA is provided by Gcn5, but the complex also contains a ubiquitin specific protease activity provided by USP22 (humans and mice) or its orthologs, nonstop (flies) and Ubp8 (yeast). USP22 was identified as part of an 11 gene 'death from cancer' stem cell signature in highly metastatic, therapy-resistant human tumors, but how over expression of USP22 contributes to a cancer stem cell phenotype, or whether these functions are related to the overall functions of the SAGA complex is not yet clear. We previously created several mutant alleles of Gcn5 to probe SAGA functions during mammalian development, and our studies indicate that the SAGA complex has important HAT-independent functions during early development. At least some of these functions are likely related to USP22, since we discovered that Gcn5 deletion results in diminished USP22 activity (Atanassov et al, 2009). We hypothesize that USP22 has important functions in embryonic stem cells and in embryo development that may be directly related to its roles in tumorigenesis. Our recent studies using USP22 gene trap alleles confirm that USP22 functions are important for normal embryo development and survival. We will further define the developmental roles of USP22 through: 1) Definition of SAGA dependent and independent USP22 functions by creating mice bearing either a floxed allele or a catalytically dead allele of USP22 2) Definition of USP22 functions in mouse ES cells before and after differentiation through both loss of function and over expression approaches and 3) Determination of genomic locations, gene targets and protein substrates of USP22 in ES cells. In the long term, these studies will provide important insights into how USP22 over expression contributes to the formation or maintenance of cancer stem cells and the development of highly aggressive tumors.